MAO Inhibitors and Their Conjugates as Therapeutics For The Treatment of Brain Cancer

ABSTRACT

A pharmaceutical composition and method for treating brain cancer are provided. The method includes administering to a patient in need thereof an effective amount of one or more compounds that include moclobemide, clorgyline, clorgyline&#39;s Near-infra-red dye Monoamine Oxidase Inhibitor (NMI), and MHI 148-clorgyline, and their salt thereof. The composition and method are particularly effective in reducing the size of glioblastomas that are temozolomide (TMZ) resistant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of application Ser. No.13/559,431, filed on Jul. 26, 2012, the entire content of which isincorporated herein by reference. Also this application claims priorityto U.S. Provisional Application Ser. No. 61/937,425, filed on Feb. 7,2014, the content of which is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Contract No.R01-MH39085 awarded by the National Institute of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to inhibition of monoamineoxidases (MAOs) and their inhibitors (MAOIs) in the treatment of braincancer.

BACKGROUND OF THE INVENTION

Glioblastoma multiforme (GBM) is the most malignant form of primarybrain tumors, with a median survival time of approximately 14 months(1). Current treatments include surgery, radiation and chemotherapy. Thechemotherapeutic agent used for GBM treatment is the DNA alkylatingagent, temozolomide (TMZ). This drug is effective in combination withsurgery and radiation or as a stand-alone chemotherapy (2).Unfortunately following treatment, tumors usually recur, and no longerrespond to TMZ. Therapy options are then very limited. Therefore, theidentification of a drug that is well-tolerated, cytotoxic for gliomasand able to cross the blood-brain-barrier would be extremely useful forthe treatment of TMZ -resistant recurrent GBM.

Monoamine oxidase A (MAO A) is a mitochondrial-bound enzyme whichcatalyzes oxidative deamination of monoamine neurotransmitters such asserotonin, norepinephrine, dopamine and produces hydrogen peroxide(H₂O₂₎, a reactive oxygen species (ROS) which predisposes cancer cellsto DNA damage, thereby promoting tumor initiation and progression.Previous studies in the inventors' lab showed that knock-down (KD) orpharmacological inhibition of MAO A in prostate cancer reduced oreliminated cancer progression (3). Clorgyline, a selective MAO Ainhibitor (MAOI) which crosses the blood-brain-barrier (BBB), is used asan anti-depressant, and causes reduced prostate cancer growth in vivo.The clorgyline conjugate NMI significantly reduced tumor growth, and isselectively cytotoxic for cancer cells in vitro and in vivo, alsocrosses the BBB, and is visualized by near-infrared imaging useful forcancer diagnosis and monitoring cancer progression.

Increased MAO A expression has previously been reported in severalcancers including prostate cancer and renal cell carcinoma (15, 16) andis down regulated in majority of human cancers, based on an ensemble ofcancer GeneChip dataset (17). Previously, the inventors showed thatelevated expressions of MAO A promoted prostate tumorigenesis, andinduced epithelial-to-mesenchymal transition (EMT) in prostate cancercells. Furthermore, inhibition of MAO A reduced the growth of LNCaP PCacells in vitro and tumor xenograft in vivo (18, 3)

SUMMARY OF THE INVENTION

One aspect of the present invention is direct to compounds andpharmaceutical compositions useful for the treatment of brain cancer andto treatment methods for treating brain cancer using the compounds andpharmaceutical compositions of the present invention.

A method of treating brain cancer according to the present inventioncomprises administering to a patient having brain cancer and in need oftreatment an effective amount of an MAO inhibitor. The brain cancer tobe treated may be a glioblastoma, and can include Glioblastomamultiforme. The brain cancer to be treated may also be TMZ resistantglioblastoma or Glioblastoma multiforme.

The MAO inhibitor is preferably selected from the group consisting of

Preferably, the MAO inhibitor is selected from the group consisting of

In a preferred embodiment, the MOA inhibitor may be covalently linked toa near infrared dye via a linker. The near infrared dye comprises apolyene functional groups, and is preferably a near infrared dye isselected from the group consisting of IR-783, IR-780, IR-786 andMHI-148, and more preferably, MHI-148.

In a preferred embodiment, the MAO inhibitor is selected from the groupconsisting of a conjugate of MHI-148 and moclobemide (MHI-moclobemide),a conjugate of MHI-148 and phenelzine, a conjugate of MHI-148 andtranylcypromine, a conjugate of MHI-148 and pargyline, a conjugate ofMHI-148 and clorgyline.

Preferred examples include:

and salts, carboxylic acids or esters thereof.

Other examples include compounds having the formula:

Another embodiment of the present invention is a compound comprising asalt of

or a carboxylic acid or ester analog thereof, and to pharmaceuticalcompositions comprised thereof and to methods and uses thereof fortreating brain cancer.

In another embodiment, the present invention is directed to compoundsand pharmaceutical compositions useful for the treatment of drugresistant brain cancer and to treatment methods for the treatment ofdrug resistant brain cancer. The methods of treating drug resistantbrain cancer according to the present invention generally includeadministering to patient in need thereof an effective amount of a MAOinhibitor.

In another embodiment, the present invention is directed to compoundsand pharmaceutical compositions useful for sensitizing TMZ resistantbrain cancer to TMZ and to treatment methods for the treatment of TMZresistant brain cancer. The methods of treating drug sensitizing TMZresistant brain cancer to TMZ treatment according to the presentinvention generally include administering to patient in need thereof aneffective amount of a MAO inhibitor. The method preferably includesconcurrently or sequentially administering an effective amount of TMZ.

The present invention shows that inhibition of monoamine oxidase A (MAOA) reduces tumor growth and increases survival of temozolomide(TMZ)-resistant gliomas. Gliomas initially respond to TMZ; but patientsusually become resistant to this drug and tumors recur. No treatment isthen available. MAO A is a mitochondrial enzyme which oxidativelydeaminates monoamine neurotransmitters, produces hydrogen peroxidecausing cell damage and cancer. Human gliomas express MAO A, whereasnormal astrocytes have no detectable MAO A activity. In vitro studiesshowed that both MAO inhibitor clorgyline and NMI, defined herein,increased TMZ sensitivity in drug-sensitive glioma cells, while inTMZ-resistant cells only NMI sensitized cells but not clorgyline. NMI(IC₅₀: 5 μM) is more effective than clorgyline (IC₅₀: 140 and 136 μM)for reducing migration in both resistant and sensitive human gliomacells. Mouse GL26 tumor implanted in MAO A KO mice exhibited increasedsurvival compared to wild type, suggesting that MAO A in themicroenvironment affects tumor growth and survival. Drug efficacystudies in orthotopic xenograft models showed that both clorgyline andNMI decreased the growth of TMZ-resistant tumors and increase survival(28%, 46%, respectively). Analysis of tumor tissues showed that MAO Ainhibitors reduced proliferation, microvessel density, and matrixmetalloproteases, and increased macrophages infiltration. In summary,the present invention has discovered an important role for MAO A inprogression of drug-sensitive and resistant gliomas, and identified MAOA inhibitors and their conjugates as important therapeutic agents fortreating drug-resistant brain tumors. The invention shows that MAO Ainhibitors are active in vitro and in vivo to reduce tumor progressionand prolong survival of patients with drug-resistant gliomas.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Officeupon request and payment of the necessary fee.

FIG. 1 shows the synthesis of NMI.

FIG. 2 shows that MAOA expression and activity increased in gliomatissues, mouse and human glioma cells. (A) Non-malignant brain andglioma (GBM) specimens were stained with anti-MAO A. Positive cells arered; nuclei are blue. (B) U251R and U251S cells were stained withanti-MAOA antibody; red staining showed positive cells for MAOAexpression. (C) MAO A catalytic activity in U251S and U251R, mouseglioma cells (GL26) and normal astrocytes was measured.

FIG. 3 evidences MAO A inhibition in GL26 cells by Clorgyline and NMI.MAO A activity was determined in GL26 cells in the presence ofincreasing concentration of clorgyline and NMI. Clorgyline inhibited MAOA activity with IC₅₀ value of 10⁻⁹M and NMI inhibited with 10⁻⁵ M.

FIG. 4 shows colony formation and migration assays were performed in (A)U251S and (B) U251R with TMZ (15 μM) and Clorgyline (10 μM) alone and incombination with TMZ for 48 h and the colonies were stained with 1%methylene blue on day 8 or 10 and counted. Migration assay was performedin (C) U251S and (D) U251 R cells treated with Clorgyline (1, 5, 10 μM)for 24 and 20 h, respectively. Error bars are ±standard error of themean (SEM) of experiments performed in triplicate. *p<0.05, t-test.

FIG. 5 shows the structure and functions of NMI on human glioma cells.(A) Structure of NMI. (B) Glioma cells treated with NMI. MitoTrackerreagent stained mitochondria. (C) Colony formation assays (a) U251S and(b) U251R treated with TMZ (15 μM) (T) and NMI (1, 5, 10 μM) (N) aloneand in combination with TMZ for 48 h; colonies were counted. (D) Effectof clorgyline and NMI on cell viability in (a) U251S and (b) U251R asmeasured by MTS assay. (E) Migration assay was performed on (a) U251Sand (b) U251 R cells treated with NMI (1, 5, 10 μM) for 24 h. Errorbars=standard error of the mean (SEM); experiments were performed intriplicate. ***p<0.0001, **p<0.005, *p<0.05, t-test.

FIG. 6 shows that Glioma progression decreased and survival timeincreased in MAOA KO mice. (A) Mouse glioma cells were implantedintracranially into WT and MAOA KO; and imaged 10 days later. Star (*)indicates mice tag numbers. (B) Bar graph shows luminescence (correlatedwith tumor size). (C) Survival of WT and MAOA KO mice (days) wereanalyzed using Kaplan-Meier plot. (D) Tumor and surrounding tissues wereassayed for MAO A activity.

FIG. 7 shows that Clorgyline inhibits glioma growth in mouse xenograftmodel. (A) Glioma cells were implanted subcutaneously; 7 days latertreatment was started: vehicle (water) (30 μl), or clorgyline (30mg/kg), daily for 21 days. Tumors were imaged on days 13, 28 and 41. (B)Bar graph showed tumor volume as calculated from imaging; p<0.05.

FIG. 8 shows that Clorgyline and NMI alone or in combination with TMZreduced tumor growth, and increased survival in the intracranial mousemodel. Athymic/nude mice were implanted intracranially; with humanglioma cells. After 7 days, animals were treated daily for 21 days with:vehicle, TMZ (1 mg/kg), clorgyline (10 mg/kg), TMZ (1 mg/kg)+clorgyline(10 mg/kg), NMI (5 mg/kg), TMZ (1 mg/kg)+NMI (5 mg/kg). TMZ treatmentwas given for only first 10 days. (A) NMI localizes to tumor. (B) Tumorimage 7 days post-implantation, at successive days after treatment. (C)Graph shows luminescence (correlated to tumor size). (D) TheKaplan-Meier survival curve; p values indicate that survival wasincreased in all treatment groups.

FIG. 9 shows that clorgyline and NMI reduced proliferation andangiogenesis and induced the innate immune response in vivo (A) Tissuesfrom vehicle, clorgyline, and NMI treated animals were immunostainedwith Ki67, MMP 9 and CD31. Red color indicates positive staining. (mag.400×). Tissue sections were (B) immunostained for F4/80, TNF-α and TGFβ.Red color indicates positive staining. (mag. 400×). (C) Quantificationof Ki67, CD31 and F4/80 in treated tissues. *p<0.05, **p<0.01 (comparedto vehicle).

FIG. 10 shows that MAO A inhibitors reduce tumor growth, MAOA activityand increase animal survival. A) mouse glioma cells were implantedintracranially: after 6 days, animals were treated daily with vehicle,TMZ (1 mg/kg), phenelzine (10 mg/kg), TMZ (1 mg/kg)+phenelzine (10mg/kg) and moclobemide (10 mg/kg). Survival was analyzed using theKaplan-Meier plot; p values indicate that all treatments significantlyincreased survival. B) MAOA activity was reduced after treatment withMAO inhibitors.

FIG. 11 shows that MAOA expression increased in glioblastoma (GBM)tissue compared to normal brain. Frozen sections of normal and glioma(GBM) brain specimens were stained with anti-MAOA antibody. Positivecells show red precipitate; hematoxylin stains (blue) nucleus. MAOAexpression increased in glioblastoma (GBM) tissue compared to normalbrain.

FIG. 12 shows that MAO A and MAO B activity in tumor-derived cell lines(LN229, U251), GSC (USC02,USC08) and in GBM but not in normal astrocytessuggests that MAOA activity is associated with cancer progression. Totalprotein from cells was collected and incubated with 10 μM of ¹⁴C-labeled serotonin at 37° C. for 20 min. Radioactivity was measured byliquid scintillation spectrophotometry.

FIGS. 13A-13C show that glioma growth is significantly decreased inMAOA/B knockout (KO) mice. A) Luciferase-labeled GL-26 glioma cells wereimplanted intracranially into WT and A/B KO C57 B/L mice and imagedafter 10 days. B) Bar graph depicts the luminescence which correlatedwith tumor size. C) Comparison of animal survival of WT and MAOA/B KOmice. Survival rate was increased by 116.6% (14 days). ***p<0.0001.

FIG. 14 shows that treatment of human glioma cells with clorgylinedecreases glioma cell migration. Glioma cells (LN229) were grown toconfluency, and then treated with mitomycin C to prevent proliferation.A “scratch” was made to clear an area of cells; then cultures weretreated with (A) vehicle or (B) clorgyline [10 μM] for 24hrs. Theresults show that clorgyline decreased the rate of migration byapproximately 50%. (C) Summary of data.

FIG. 15 shows that treatment with the MAO A inhibitor, clorgyline,induces glioma stem cell cytotoxicity. (A) GSC were treated withclorgyline for 96 hrs and assayed using sphere formation; (B) GSC weretreated with clorgyline and TMZ for 72 hrs, clorgyline was added againfor another 48 hrs and assayed (MTT). Results show that clorgyline iscytotoxic for glioma stem cells at 5 and 10 μM. Furthermore, clorgylinein combination with TMZ exhibits a 50% increase in cytotoxicity.

FIG. 16 shows that MHI-Clorgyline Reduces Colony Formation inTemozolomide-Resistanl Glioma Cells. U251 TMZ resistant (R) were platedin 6-well plates and treated with MAOA inhibitors and TMZ for 48 hrs.MHI-Clorgyline reduces the colony formation (CFA) rate by 65% as compareto vehicle. Combined treatment of TMZ and MHI-Clorgyline sensitizesTMZ-resistant glioma cells to TMZ, resulting in reduced colony formationby 85%. CFA is a measure of cell death; the fewer colonies the more celldeath.

FIG. 17 shows that the administration of MHI-Clorgyline alone or incombination with temozolomide (TMZ) increases survival in animalsbearing intracranial TMZ-resistant gliomas. Athymic/nude mice wereimplanted intracranially with U251-TMZ-resistant glioma cells. After 7days, animals were separated into the following groups and treateddaily: vehicle, TMZ (1 mg/kg), Clorgyline (10 mg/kg), MHI-Clorgyline (5mg/kg), or TMZ (1 mg/kg)+MHI-Clorgyline (5 mg/kg). After 21 days,treatment was stopped; survival was documented. The results showed thatMHI-Clorgyline-treated animals survived 50% longer than the vehicletreated animals (p<0.0001), and TMZ+MHI-Clorgyline-treated mice survived70% longer than vehicle (p<0.00001). Inhibition of MAOA usingMHI-Clorgyline alone or in combination with TMZ is effective indecreasing glioma tumor progression. MHI-Clorgyline sensitizesTMZ-resistant cells to TMZ.

FIG. 18. Prostate cancer and glioma have MAO A activity and can betreated with MAO I and MHI-clorgyline. Pancreatic cancer and lymphoma donot have MAO A activity, thus cannot be treated by clorgyline andMHI-clorgyline.

FIG. 19. A) Presence of MAO A activity in gliomas stem cells (GSC) andB) treatment with the clorgyline and MHI-clorgyline, induces glioma stemcell cytotoxicity. (A) GSC, USC08 and USC02, showed MAOA activity. MAOAactivity was determined by a radioactivity assay. Cell homogenate wereincubated with 10 μM of ¹⁴C-labeled serotonin at 37° C. for 20 min. Theproduct of MAOA catalyzed reaction, 5-HIAA, was extracted and theradioactivity was determined by liquid scintillation spectrophotometry.B) USC08 and USC02 stem cells were treated with Clorgyline andMHI-clorgyline with increasing concentration and sphere formation wasmeasured. Clorgyline and MHI-clorgyline induce the stem cellcytotoxicity.

DETAILED DESCRIPTION OF THE INVENTION

Pharmaceutical compositions suitable use in connection with the presentinvention are generally prepared by mixing the active ingredient havingthe desired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate and otherorganic acids; antioxida.nts including ascorbic acid; low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, ordextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™,PLURONICS™ or PEG.

The formulations to be used for in vivo administration should generallybe sterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution.

Definitions

Patients who may benefit from or are in need to the treatment methods ofthe present invention are those who have been diagnosed with braincancer.

As used herein, the phrase “treating brain cancer” may include having atleast one of the following effects: to inhibit the inhibit the formationor spread of primary tumors, macrometastases or micrometastases,decrease the size of macrometastases or to ameliorate or alleviate oneor more symptoms of the disease caused by the brain cancer. An“effective amount” of the pharmaceutical compositions of the presentinvention is an amount sufficient to carry out a specifically statedpurpose. An “effective amount” may be determined empirically and in aroutine manners in relation to the stated purpose. Inhibiting orreducing the formation, size or spread of macrometastases ormicrometastases may be shown by comparing to untreated controls.

Disclosed herein are results showing that MAO A mediates gliomaprogression, and inhibiting MAO A activity can decrease glioma growth,thereby increasing survival. The data presented here demonstrated thatthe MAO inhibitors moclobemide, clorgyline and its derivative NMI areactive in decreasing tumor growth. It was demonstrated that tissuespecimens from GBM patients overexpress MAO A as compared to non-tumorbrain tissues. The effects of MAO A inhibitors, currently in use asanti-depressants were examined for the treatment of brain cancer,including drug-resistant gliomas.

The results presented herein demonstrate MAO A's role in brain tumorgrowth, and tumor microenvironment. One big challenge in the treatmentof gliomas is tumor recurrence, and these recurrent tumors are commonlyTMZ -resistant. One important aspect of the present invention is thatMAO A inhibitors and their conjugates reduce glioma growth andprogression in TMZ-resistant, a result of significant clinicalsignificance.

One aspect of the present invention is directed to a compound andpharmaceutical composition used for treating treating brain cancer,including malignant gliomas, and including Glioblastoma multiforme(GBM). Another aspect is directed to methods of treating the malignantglioma (such as GBM) using the compositions. Compositions in accordancewith this aspect of the invention will generally include an MAOinhibitor (i.e. an active agent capable of inhibiting MAO activity); anda physiologically suitable carrier. In one aspect, a method for treatinga malignant glioma comprises contacting the glioma with an effectiveamount of the MAO inhibitor. The method of treating a patient with amalignant glioma or GBM in connection with this aspect of the presentinvention comprises administering to a patient in need thereof aeffective amount of the MAO inhibitor. The MAO inhibitor may be anano-conjugate with a NIR dye conjugated to a MAO inhibitor.

A second aspect of the present invention is directed to a pharmaceuticalcomposition useful for to reduce GBM progression, and methods of slowingthe progression of GBM using the compositions. Slowing the progression,as used herein, includes halting the progression, slowing theprogression, or slowing the rate of increase of progression of at leastone characteristic of GBM tumors. Compositions in accordance with thisaspect of the invention will generally include an MAO inhibitor (i.e. anactive agent capable of inhibiting MAO activity); and a physiologicallysuitable carrier. The method of reducing GBM progression in connectionwith this aspect of the present invention comprises administering to apatient in need thereof a effective amount of the MAO inhibitor. The MAOinhibitor may be a nano-conjugate with a NIR dye conjugated to a MAOinhibitor.

A third aspect of the present invention is directed to a pharmaceuticalcomposition useful for decreasing GBM cell migration and invasion, andmethods of decreasing GBM cell migration and invasion the compositions.Compositions in accordance with this aspect of the invention willgenerally include an MAO inhibitor (i.e. an active agent capable ofinhibiting MAO activity); and a physiologically suitable carrier. Themethod of decreasing GBM cell migration and invasion in connection withthis aspect of the present invention comprises administering to apatient in need thereof a effective amount of the MAO inhibitor. The MAOinhibitor may be a nano-conjugate with a NIR dye conjugated to a MAOinhibitor.

A fourth aspect of the present invention is directed to a pharmaceuticalcomposition useful for decreasing glioma stem cell activity, and methodsof decreasing glioma stem cell activity using the compositions.Compositions in accordance with this aspect of the invention willgenerally include an MAO inhibitor (i.e. an active agent capable ofinhibiting MAO activity); and a physiologically suitable carrier. Themethod of decreasing glioma stem cell activity in connection with thisaspect of the present invention comprises administering to a patient inneed thereof a effective amount of the MAO inhibitor. The MAO inhibitormay be a nano-conjugate with a NIR dye conjugated to a MAO inhibitor.

A fifth aspect of the present invention is directed to a pharmaceuticalcomposition useful for sensitizing TMZ-resistant gliomas, including TMZresistant GBM, to TMZ, thereby making these TMZ-resistant cellssensitive to this drug, and methods of sensitizing TMZ-resistantresistant gliomas, including TMA resistant GBM, using the compositions.Compositions in accordance with this aspect of the invention willgenerally include an MAO inhibitor (i.e. an active agent capable ofinhibiting MAO activity); and a physiologically suitable carrier. Themethod of sensitizing TMZ-resistant GBM to TMZ in connection with thisaspect of the present invention comprises administering to a patient inneed thereof a effective amount of the MAO inhibitor, sensitizingTMZ-resistant GBM to TMZ, thereby making these TMZ-resistant whereby theamount is effective to sensitive the TMA-resistant GBM cells to TMZ. TheMAO inhibitor may be a conjugate with a NIR dye conjugated to a MAOinhibitor.

A sixth aspect of the present invention of the present invention is acombination treatment for the treatment of malignant gliomas, includingGBM, comprising the administration of a combination of TMZ and an MAOinhibitor. TMZ and the MAO inhibitor may be administered concurrently.Conversely, the MAO inhibitor and TMZ may be administered sequentially,with the MAO inhibitor preferably administered first.

Suitable MAO inhibitors, including suitable nano-conjugates thereof,include those described in U.S. patent application Ser. No. 13/353,094filed Jul. 26, 2013, which is incorporated herein by references in itsentirety. Suitable MAO inhibitor, and nano-conjugates thereof, may besynthesized according to the methods described in U.S. patentapplication Ser. No. 13/353,094.

In one embodiment, the MAO inhibitor used in connection with the presentinvention are known in the art. Exemplary MAO inhibitor may include, butnot limited to moclobemide, phenelzine, tranylcypromine, pargyline, andclorgyline. Nucleic acids capable of inhibiting, down-regulating orsilencing the expression of MAO may also be advantageously used.Exemplary nucleic acid MAO inhibitors may include siRNA, shRNA,antisense, or any other type of nucleic acid-based gene silencing agentscommonly known in the art, such as decoys, ribozymes, and aptamers. Suchpreferred embodiments can be used, either alone or in combination withthe described herein pharmaceutical compositions as cancer therapeutics.

Suitable MAO inhibitors include:

-   -   and compounds 11-14 as shown below:

and salts thereof.

As set forth in U.S. patent application Ser. No. 13/353,094 filed Jul.26, 2013, the MAO inhibitors may linked to a near IR dye (NIR) to form aconjugate capable of, for instance, preferentially or selectivelytargeting cancer cells. Conjugates in accordance with this aspect of theinvention will generally have an NIR dye nanoparticle conjugated to anMAO inhibitor. Exemplary NIR dyes may include conjugated polyenefunctional groups, such as one found in IR-783, IR-780, IR-786, andMHI-148 but are not limited thereto. Exemplary MAO inhibitors includePhenelzine, Tranylcypromine enantiomers, Pargyline, Rasagiline,D-deprenyl, L-deprenyl, and compounds 11-14 as shown below:

and salts thereof.

Conjugation of the NIR dye to the MAO inhibitor may be achieved by anysuitable chemical means known in the art.

In one preferred embodiment, exemplary conjugates of the presentinvention will generally have at least two functional groups: an MAOinhibitor attached to a light emissive element (e.g. NIR dyenanoparticle) via a linker containing at least one C and two H atoms.Preferably, at least two unsaturated structures containing oneunsaturated double or triple bond are linked via a backbone chain of1-3, 1-5, or 1-15 atoms to one heterocycle.

An exemplary linker is one having the following general formula:

wherein M₁ is O or S; and wherein at least two of X, Y, and Zparticipate in bonds to unsaturated and/or aromatic groups A and B (notshown) which proceed through additional carbon, oxygen or nitrogenatoms. Any of X, Y, and Z not participating in a bond to group A or B issubstituted with hydrogen or lower aliphatic group, such as C₁-C₆ alkyl.

For example, a conjugate in accordance with embodiments of the inventionmay be one having the following formula:

In another embodiment, X and Y are as above and Z is selected from thegroup consisting

wherein the covalent link is attached to the aromatic ring. Thiscompound is herein referred to as MHI-moclobemide, a MAO-A specificreversible inhibitor.

In another embodiment, X and Y are same as above, and Z is

wherein the covalent bond is also attached to the aromatic ring. Thiscompound is herein referred to as MHI-phenelzine, a MAO-A and -Binhibitor.

In still another embodiment, X and Y are same as above, and Z is(±)-trans-2-phenylcyclopropan-1-amine having the formula:

wherein covalent attachment is through the aromatic ring. This compoundis herein referred to as MHI-tranylcypromine, which is a MAO-A and -Binhibitor.

In still another embodiment, X and Y are same as above, and Z isN-Benzyl-N-methylprop-2-yn-1-amine, having the following formula:

wherein covalent linkage is attached to the nitrogen as indicated by thecurly line. This compound is herein referred to as MHI-pargyline, aMAO-A and -B inhibitor with a preference for MAO-B.

In a preferred embodiment, Y is S; X is a group having the followingformula:

and Z is a group having the following formula:

This compound is referred to herein as MHI-clorgyline, which is a MAO-Aspecific irreversible inhibitor.

In yet another embodiment, X and Y are same as above, Z is one selectedfrom the following:

wherein covalent linkage is attached to the aromatic rings. This groupof compounds is collectively referred to herein as MHI-MAOIs.

In another embodiment, an MAO inhibitor NIR dye conjugate has theformula:

In one preferred embodiment and to improve the targeting of MAOinhibitors specifically to tumors, the inventors designed an exemplaryNIR dye-MAOA inhibitor conjugate, NMI, evidencing that NIR dye-MAOAconjugates specifically target the mitochondria of cancer cells withoutaffecting normal cells (19). NMI is shown in an anionic form as follows:

As used herein, NMI includes salt forms of the anion or the carboxylicacid an ester analog of the salt.

Suitable pharmaceutically acceptable base addition salts of compounds ofthe invention include, for example, metallic salts including alkalimetal, alkaline earth metal and transition metal salts such as, forexample, calcium, magnesium, potassium, sodium and zinc salts.Pharmaceutically acceptable base addition salts also include organicsalts made from basic amines such as, for example,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. Examples ofpharmaceutically unacceptable base addition salts include lithium saltsand cyanate salts. Although pharmaceutically unacceptable salts are notgenerally useful as medicaments, such salts may be useful, for exampleas intermediates in the synthesis of compounds, for example in theirpurification by recrystallization. All of these salts may be prepared byconventional means from the corresponding compound by reacting, forexample, the appropriate acid or base with the compound. The term“pharmaceutically acceptable salts” refers to nontoxic inorganic ororganic acid and/or base addition salts, see, for example, Lit et al.,Salt Selection for Basic Drugs (1986), Int J. Pharm., 33, 201-217,incorporated by reference herein

NMI comprises clorgyline, an irreversible MAOA inhibitor, conjugated tothe NIR dye MHI-148 by an amide bond via a linker. This bifunctionalsystem has the NIR imaging capability for diagnosis as well therapeuticactivity to specifically target cancer.

The inventors have, for example, examined the efficacy of clorgyline andMHI-clorgyline on tumor growth both in vitro and in vivo systems. Theinventors have shown that these MAO A inhibitors are cytotoxic for GBMcells and glioma stem cells. The inventors have demonstrated that, inthe orthotopic in vivo mouse models, MAO inhibitors reduce brain tumorprogression of TMZ

resistant GBM. Furthermore, MAO inhibition affects both the glioma cellsand the microenvironment of the tumor.

As shown herein, NMI is targeted specifically to mitochondria in humanglioma cell and inhibited MAOA activity. NMI has better efficacy thanclorgyline in both in vitro and in vivo assays. NMI inhibited colonyformation for drug-sensitive and drug-resistant glioma, and furthersensitized the sensitive and resistant glioma cells to TMZ treatment.This indicates that the mechanism(s) by which MAO inhibitors affectglioma cells are independent of TMZ-mediated activity.

These results demonstrate that NMI is 30 times more effective ininducing cytotoxicity in glioma cells as compared to clorgyline,suggesting that this increase in cell death may be the result, at leastin part, of the higher accumulation of the NIR conjugate in the cancercells. The inventors also explored the ability of clorgyline and NMI toaffect tumor cell migration a critical characteristic of recurrentglioma and found that NMI significantly inhibited the migration of humanglioma cells, however clorgyline did not show any significant efficacy.This observation is also consistent with the concept that NMI iseffective in depositing high doses of the MAO inhibitor in the tumorcells, and that decreasing MAO A correlates with decreased tumor growth.Thus targeting the tumor with the NIR moiety to produce NMI is a farmore effective inhibitor of tumor growth.

Cancer cells are usually under persistent pressure of a complex tumormicroenvironment, which includes hypoxia, acidosis, oxidative stress andseveral other factors (21, 22). High oxidative stress results inelevated expression of reactive oxygen species (ROS) such as, hydrogenperoxide (23). MAOA, when catalyzing oxidative reactions produceshydrogen peroxide as a by-product that can be further converted intoother species of ROS (24). The inventors' results showed reduced tumorgrowth and increased survival in MAO A KO mice as compared to wildtype.Interestingly, it was found that MAO A activity in the tumor andsurrounding tissue was lower compared to wildtype, suggesting that MAO Ain the microenvironment may affect glioma growth and survival. Inspiredby these results the inventors explored the potential of other MAOinhibitors on tumor growth. The intracranial xenograft in vivo mousemodel demonstrated increased survival response with moclobemide and incombination with phenelzine and TMZ. These data showed that a variety ofMAO inhibitors were effective in decreasing tumor growth. The in vivostudies using intracranial xenograft mouse models showed that clorgyline(10 mg/kg) and NMI (5 mg/kg) both reduced the rate of tumor growth andincreased survival. The results also showed that the combination of TMZand clorgyline or NMI exhibited a significant additive effect forsurvival, compared to each drug alone. NMI (5 mg/kg) showed significantinhibitory efficacy on tumor xenograft growth, and also showed selectivelocalization to the tumor. These data indicated that NMI was moreeffective than clorgyline alone, and likely to be more useful especiallyfor TMZ-resistant gliomas.

To determine the potential mechanisms of the in vivo effects of MAO Ainhibitors, tumor tissues from treated animals were examined for severalcharacteristics including microvessel density. The data indicate thatboth clorgyline and NMI decrease blood vessel growth. The reasons forthis decrease are not clear. Clorgyline and NMI do not appear to affectnormal blood vessels, since the blood vessel density in the adjacentbrain parenchyma did not appear to exhibit abnormal density or vascularstructure. Thus the MAO A inhibitors specifically affect the tumorvasculature. Since the microenvironment of the tumor vasculature oftenexpresses high levels of vascular endothelial growth factor (VEGF), andbasic fibroblast growth factor (bFGF), as well as low levels ofthrombospondin-1 (TSP-1), compared to normal brain (25, 26), there isthe possibility that MAO A inhibitors may also regulate these growthfactors.

Tumor tissues from animal treated with MAO inhibitors exhibited highnumbers of macrophages, as compared to control tissues. There isconsiderable evidence that the immune system, especially macrophages, isimportant in regulating tumor progression (10). Proinflammatorymacrophages generally decrease tumor growth, while immune suppressive,anti-inflammatory macrophages maintain or enhance tumor growth (27). Acharacteristic of proinflammatory macrophages is the production ofTNF-α. Our immunohistochemistry staining data showed that NMI—treatedtissues demonstrated a significant increase in TNF-α, indicating thatthe macrophages present here were likely to be proinflammatory cells.Thus MAO inhibitors regulated the activity of macrophages as well as theactivity of glioma cells.

In conclusion, the inventors have demonstrated that in vivo MAO Ainhibitors reduced glioma growth and increase survival. These effects ofMAO inhibitors may be the result of reduced proliferation, increasedcytotoxicity, and/or decreased invasion of tumor cells. MAO A inhibitorsmay also function by regulating macrophage activity and/or decreasingblood vessel density within the tumor. It is likely that MAO Ainhibitors performs several different functions, thereby enabling theseinhibitors to be effective against drug-resistant gliomas, Thustargeting MAO A for the treatment of glioma is an effective approach tothe treatment of recurrent brain tumors.

Experimental A. Material and Methods

Cell cultures. The human glioma cell line U251 was obtained fromAmerican Type Culture Collection; the TMZ-resistant human glioma cells,U251R, was derived as previously described (4). Mouse glioma cell lineGL26 was a gift from Dr. Linda Liau, UCLA. All glioma cell lines werecultured in 10% fetal calf serum (FCS) in Dulbecco's Modified Eagle'sMedia (Life technologies, Carlsbad, Calif.) supplemented with 100 U/mLpenicillin and 0.1 mg/mL streptomycin in a humidified incubator at 37°C. and 5% CO₂.

MAO A catalytic activity assay. MAOA catalytic activity was determinedas described previously (5). Briefly, cell or tissue homogenates wereincubated with 1 mM ¹⁴C-5-hydroxytryptamine (5-HT) in assay buffer.Reaction products were extracted and radioactivity was determined byliquid scintillation spectroscopy. For inhibition activity assays, cellswere pre-incubated with various compounds at increasing concentrations,for 20 minutes at 37° C. followed by the addition of ¹⁴C labeled 5-HT at37° C. for 20 minutes.

Laser-scanning confocal microscopy. Cells were plated in glass bottommicroscopy dishes (MatTek) (30,000 cells/400 μl), in standard medium,for 24 hours. Cells were treated with NMI (1 and 5 μM), MitotrackerGreen (200 nM) (Life Technologies, Carlsbad, Calif.) and DAPI (1×); andincubated for 3 hours. Imaging was performed on a Zeiss LSM 510 invertedlaser-scanning confocal microscope. Excitation wavelengths were set atλ_(max)=790 nm Chameleon (DAPI, blue excitation), 488 nm (MitotrackerGreen, green-yellow excitation) and 633 nm (NMI, red excitation). Thedata were acquired in a multi-track mode. Images were taken usingpinholes of 130-200 μM.

Colony forming assay. Glioma cells were seeded in triplicates, thentreated with clorgyline, NMI and TMZ for 48 hours. The medium was thenremoved and replaced with fresh medium (without drugs). Cells wereincubated for an additional 8 to 10 days; colonies were visualized bystaining with 1% methylene blue and quantified.

MTS assay. Glioma cells were seeded in quadruplicates, and clorgylineand NMI added for 48 hours. Viability was determined as per manufacturesinstructions (Promega, Madison, Wis.); and calculated relative tountreated control cells. Data was plotted using SigmaPlot 12.0.

Migration assay. Cells treated with mitomycin C (10 μg/ml) (SigmaAldrich, St. Louis, Mo.) for 2 hours; a scratch was made using 200 μLsterile tip. Cells were then treated with drug: and photographed at 0hours and 20-24 hours. Migration was quantified by measuring the areacovered by control versus treated groups using ImageJ.

In vivo studies. All animal protocols were approved by the InternationalAnimal Care and Use Committee of USC. Intracranial implantation wasperformed as previously described (6). For MAOA KO studies (7), C57bl/6mice were used. 2×10⁴ luciferase labeled GL-26 mouse glioma cells wereimplanted intracranially into wild type (WT) (n=4) and MAOA KO (n=4)mice; and imaged on days 6 and 10 post implantation. For treatmentstudies, tumors cells were implanted and after 6 days treatment wereinitiated. All compounds were dissolved in 10% DMSO+45% glycerol+45%ethanol and injected subcutaneously; TMZ was given by gavage. Animalswere treated daily until death, and survival was recorded.

For the xenograft model, athymic (nu/nu) mice were used.Luciferase-positive (2×10⁵) TMZ-resistant human glioma cells (U251R)were injected intracranially and imaged after 7 days. Daily (21 days)treatments began after tumors were visible; TMZ was administered for thefirst 10 days only.

In subcutaneous xenograft studies, 5×10⁵ luciferase-positive cells wereinjected subcutaneously into athymic mice. Drugs were administered dailyfor 21 days; and tumor size was monitored. Control animals were treatedwith vehicle.

Immunohistochemistry (IHC). Frozen tissues were fixed in acetone. IHCwas performed as described previously (8). The following primaryantibodies were used: F4/80, TGF-β, TNF-α (Abcam, Cambridge, Mass.),Ki67 (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.), CD31 (BDBiosciences, San Jose, Calif.), MMP 9 MAO A (Santa Cruz BiotechnologyInc., Santa Cruz) and biotinylated secondary antibodies (VectorLaboratories, Berlingame, Calif.). Controls included no primaryantibody. Images were analyzed using Image J software.

Statistical analysis. All parametric data were analyzed using thetwo-tailed student t-test to calculate the significance values. Aprobability value (p)<0.05 was considered statistically significant.

B. Synthesis of NMI General Synthetic Methods

All reagents and solvents were obtained from commercial sources and wereused as received unless otherwise stated. All reactions involvingmoisture-sensitive reagents were conducted under argon atmosphere withanhydrous solvents and flame-dried glassware. Hygroscopic liquids weretransferred via a syringe and were introduced into reaction vesselsthrough rubber septa. Reaction product solutions were concentrated usinga rotary evaporator at 30-150 mm Hg. Column chromatography was performedon silica gel (230-400 mesh) using reagent grade solvents. Analyticalthin-layer chromatography (TLC) was performed on glass-backed,pre-coated plates (0.25 mm, silica gel 60, F-254, EM Science).Analytical HPLC were performed on Microsorb-MV C₈ reverse-phase column(250×4.6 mm, Varian) using Shimadzu LC-10A VP pump and Shimadzu SPD 10AVP UV-vis variable-wavelength detector. Preparative HPLC purificationswere carried out with C₈ reverse phase preparative column (GraceDavison). The flow rate for preparative reverse-phase HPLC was 4 mL/min.In all cases, 5%-95% gradients of acetonitrile in 0.1% aqueoustrifluoroacetic acid (TFA) were used as eluents. Water (18 MΩ) wasobtained from a Barnstead water purification system, and all bufferswere 0.2 μm filtered. Nuclear magnetic resonance (NMR) spectra werecollected on instruments in the indicated solvents. The identity andpurity of each intermediate and the final product were confirmed by ¹HNMR (Varian Mercury 400 MHz) and mass spectrometry (Agilent 6520time-of-flight system). An overview of the synthesis of NMI is disclosedin FIG. 1.

t-Butyl (3-bromopropyl)carbamate (1)

To a 100-mL round-bottom flask equipped with a magnetic stirrer wasadded 3-bromopropylamine hydrobromide (6.57 g, 30 mmol, 1.0 eq.) anddichloromethane (160 mL). To the resultant solution was addeddi-tert-butyl dicarbonate (6.54 g, 30 mmol, 1.0 eq.) in dichloromethane(110 mL), followed by triethylamine (4.8 mL, 34.5 mmol, 1.15 eq.). Thesolution was stirred at room temperature for 95 minutes. The reactionwas washed twice with saturated sodium bicarbonate (70 mL, 40 mL) andonce with saturated sodium chloride (100 mL). The organic phase wasdried over sodium sulfate, filtered, and solvent removed in vacuo toyield 1 (6.91 g, 97% yield).

¹H NMR (400 MHz, CDCl₃) δ: 4.67 (bs, 1 H), 3.43 (t, J=6 Hz, 2H), 3.26(m, 2 H), 2.04 (m, 2 H), 1.43 (s, 9 H).

t-Butyl 3-(2,4-dichlorophenoxy)propylcarbamate (3)

To a 20-mL scintillation vial equipped with a magnetic stirrer was added2,4-dichlorophenol (2, 237 mg, 1.45 mmol, 1.0 eq.) followed by1,2-dichloroethane (5 mL). To the resultant solution was added 1 (346mg, 1.45 mmol, 1.0 eq.), tetrabutylammonium chloride (44.2 mg, 0.159mmol, 0.11 eq.), followed by potassium iodide (24.1 mg, 0.145 mmol, 1.0eq). NaOH (5 mL of 10%) was added and the mixture was stirred at 80° C.for 1 h. The biphasic mixture was partitioned and the aqueous layer wasextracted twice with DCM (7 mL). The organic phases were pooled, driedover magnesium sulfate, filtered, and then concentrated. The material(512.7 mg) was then columned over silica gel (2.5″×1″, height×width)eluting with 100 mL 10% ethyl acetate in hexanes, 100 mL 15% ethylacetate in hexanes, and 100 mL 25% ethyl acetate. Product 3 elutes in15% ethyl acetate fractions. Yield 310 mg (67%). ¹H NMR (400 MHz, CDCl₃)δ: 7.32 (d, J=2.4 Hz, 1 H), 7.14 (dd, J₁=8.8 Hz, J₂=2.4, 1 H), 6.80 (d,J₁=5.2 Hz, 1 H), 5.18 (bs, 1 H), 4.04 (t, J=6 Hz, 2 H), 3.34 (m, 2 H),2.00 (m, 2 H), 1.41 (s, 9 H).

t-Butyl 3-(2,4-dichlorophenoxy)propyl(prop-2-ynyl)carbamate (4)

To a 20-mL scintillation vial was added 3 (500 mg, 1.57 mmol, 1.0 eq)followed by sodium hydride (63 mg, 1.57 mmol, 1.0 eq.). The resultingsolution was stirred for 15min with venting to atmosphere, followed bythe addition of propargyl bromide (470 μL of 80% solution in toluene(w/v), 3.14 mmol, 2.0 eq.). Note: the solution changes color fromyellowish to brown upon addition of propargyl bromide. The reaction wasstirred at room temperature for 4 h and solvent was removed via anairstream and the residue was dried in vacuo. The residue wasreconstituted in 5 mL DCM and 1.65 g of celite-545 was added. Themixture was then evaporated to dryness. This material was loaded onto a5″×1″ (height×width) plug of SiO₂ equilibrated with hexanes and productwas eluted with 100 mL 2% ethyl acetate in hexanes, 200 mL 5% ethylacetate in hexanes, and 200 mL 30% ethyl acetate in hexanes. Fractionscontaining product were pooled (product elutes between 5% and 30% ethylacetate in hexanes). Yield 292.6 mg of 4 (52% yield) ¹H NMR (400 MHz,CDCl₃) δ: 7.35 (d, J=2.8 Hz, 1 H), 7.16 (dd, J₁=9.2 Hz, J₂=2.8, 1 H),6.82 (d, J₁=8.8 Hz, 1 H), 5.18 (bs, 1 H), 4.05 (m, 4 H), 3.55 (t, J=7.2Hz, 2 H), 2.18 (t, J=2.4 Hz, 2 H), 2.00 (m, 2 H), 1.43 (s, 9 H).

N-(3-(2,4-dichlorophenoxy)propyl)prop-2-yn-1-aminium trifluoroacetate

To a solution of tent-butyl3-(2,4-dichlorophenoxy)propyl(prop-2-ynyl)carbamate 4 (147.6 mg, 412μmol) in 4 mL DCM in a 20 mL-scintillation vial equipped with a stir barwas added 1 mL TFA at room temperature while stirring. After 15 min,HPLC (5-95% B (A=0.05% aqueous TFA; B=acetonitrile) over 20 min, 0.8mL/min) indicated completion of the reaction. The solvents were removedunder reduced pressure. The residue was co-evaporated with ACN times andthen used in the next step without further purification.

t-Butyl (3-oxopropyl)carbamate (5)

To a 100 mL round bottom flask was added t-butyl(3-hydroxypropyl)carbamate (1.8 g, 10.3 mmol, 1.0 eq.) followed by 35 mLof CHCl₃ and 35 mL of 0.5M NaHCO₃/0.05M K₂CO₃. To this mixture was addedtetrabutylammonium chloride (288 mg, 1.04 mmol, 0.1 eq.), TEMPO (162 mg,1.04 mmol, 0.1 eq.), and N-chlorosuccinimide (2.1 g, 15.7 mmol, 1.53eq.) with vigorous stirring. After three hours the phases wereseparated, the organic layer was dried, and solvent was removed to givea viscous orange oil. This material was subjected to columnchromatography over silica gel using a 4″×1″ (height×width) plug of SiO₂and eluting with 75 mL hexanes, 100 mL 20% ethyl acetate in hexanes, 100mL 30% ethyl acetate in hexanes, 50 mL ethyl acetate. Yield 732.8 mg(41%).

t-Butyl(3-((3-(2,4-dichlorophenoxy)propyl)(prop-2-yn-1-yl)amino)propyl)carbamate(6)

To 100-mL round bottom flask equipped with a stir bar was addedN-(3-(2,4-dichlorophenoxy)propyl)prop-2-yn-1-aminium trifluoroacetate 4(1.25 g, 3.36 mmol, 1.0 eq.) in 25 mL 1,2-DCE. Next, 5 (0.641 mg, 3.4mmol, 1.1 eq.) was added followed by DIPEA (584 μL, 3.36 mmol, 1.0 eq),acetic acid (330 μL, 5.81 mmol, 1.73 eq.), and sodiumtriacetoxyborohydride (1.14 g, 5.38 mmol, 1.6 eq.). The reaction wasstirred for 5.5 h until approximately 85% conversion was reached, atwhich point the organic layer was washed twice with sodium bicarbonate(100 mL), dried over anhydrous MgSO₄, filtered, and solvent was removedin vacuo to give 1.12 g of crude 6. This material was used in the nextstep without further purification.

N1-(3-(2,4-dichlorophenoxy)propyl)-N1-(prop-2-yn-1-yl)propane-1,3-diaminium2,2,2-trifluoroacetate (7)

A solution ofN-(3-(2,4-dichlorophenoxy)propyl)-N-(prop-2-yn-1-yl)propane-1,3-diaminiumchloride 7 (16 mg, 38.5 μmol) in 10 mL ethyl acetate containing 248 mgof HCl was stirred mechanically. The resulting reaction was stirred atroom temperature for 80 min before reaction completion was confirmed byLC/MS. The product was evaporated under reduced pressure and used in thenext step without further purification.

NMI

To a 20-mL scintillation vial was added MHI-148 dye 8 (5.79 mg, 8.47μmol) followed by DMF (500 μL), DIPEA (1.1 μL, 8.47 μmol, 1.0 eq.), andHBtU (3.2 mg, 8.47 μmol, 1.0 eq.). The mixture was stirred for 10 min atroom temperature after which time 7 (4.6 mg, 8.47 μmol, 1.0 eq.) andDIPEA (2.2 μL, 16.94 μmol, 2.0 eq.) in DMF (500 μL) were added. Thereaction was covered in foil, and stirred overnight at room temperature.The DMF was evaporated by airstream and the residue was purified onpreparative silica gel plates using DCM/isopropanol 1:1 as an eluent.

Yield of NMI 1.2 mg (14%). ¹H NMR (400 MHz, CDCl₃): δ 1.49 (m, 2H,γ-CH₂(COOH)), 1.56 (m, 2H, γ-CH₂(CONHCH₂—)), 1.67 (s, 6H,CH₃), 1.71 (s,6H,CH₃), 1.80 (m, 2H, β-CH₂), 1.82 (m, 2H, β-CH₂), 1.86 (m, 2H, δ-CH₂),1.88 (m, 2H, δ-CH₂), 1.94 (s, 2H, O—CH₂CH₂CH₂N—), 1.97 (s, 2H,NH—CH₂CH₂CH₂N—), 2.09 (s, 2H, CH₂), 2.56-2.57 (m, 4H, α-CH₂), 2.68-2.70(m, 4H, CH₂NCH₂), 2.71 (s, 2H, CH₂C═), 2.75 (s, 2H, CH₂C═), 3.38 (m, 2H,—NHCH₂), 4.04 (t, 1H, HCΞC—), 4.05 (t, 4H, N—CH₂), 4.05 (t, 2H, O—CH₂),4.07 (t, 2H, CH₂CΞ), 4.57 (bs, 1H, NH—CH₂CH₂), 6.04 (d, 1H, CH═CH), 6.32(d, 1H, CH═CH), 6.85 (d, 1H, Ar—H), 7.05 (d, 1H, Ar—H), 7.17-7.42 (m,9H, Ar—H), 8.28 (d, 1H, CH═CH), 8.40 (d, 1H, CH═CH). HRMS calcd. forC₅₇H₆₉Cl₃N₃O₄S C₅₇H₇₀Cl₃N₄O₄ m/z 979.4457; observed m/z 979.4463.

Synthesis of MHI-148-Clorgline Conjugate Synthesis of S-3-bromopropylethanethioate

A 250 mL three-neck round-bottom flask equipped with a thermocouple in aglass sleeve, a magnetic stirrer, a vigreux column with an Argon inlet(middle stem) and a sleeved rubber septum stopper was assembled anddried with a heat gun under flow of Ar. Approximately 110-120 mL ofanhydrous DMF was added via cannula under Ar. AcSK (11.68 g, 102.3 mmol)was added by portions into the flask while cooled with ice-MeOH bath.The reaction went on for 7 h at about −10° C. The ice-MeOH bath wasremoved after quenching the reaction by adding 165 mL water. Thereaction mixture was partitioned with 300 mL MTBE and 700 mL water. Thewater layer was washed by 200 mL MTBE. The MTBE layers were washedsequentially with water, saline and NaHCO₃, dried by MgSO₄, filtered andevaporated. Yield 98.7% (19.1 g).

Synthesis ofS-3-((3-(2,4-dichlorophenoxy)propyl)(prop-2-ynyl)amino)propylethanethioate

To a solution of N-(3-(2,4-dichlorophenoxy)propyl)prop-2-yn-1-aminium2,2,2-trifluoroacetate (2.14 mg, 0.05 mmol) in 100 μL ACN in a 5 mL vialequipped with a stir bar, 12.1 mg (0.09 mmol) of K₂CO₃ and 142.4 mg(0.720 mmol) S-3-bromopropyl ethanethioate was added. The mixture wasstirred while heated to 50° C. in an oil bath. TLC was performed (silicagel, MTBE:hexane=9:1 to detect starting material and MTBE:hexane=1:9 todetect the consumption of the thioacetate reagent) to follow the processof the reaction. To a solution ofN-(3-(2,4-dichlorophenoxy)propyl)prop-2-yn-1-aminium2,2,2-trifluoroacetate (17.1 mg, 0.05 mmol) in 800 μL ACN in a 20 mLvial equipped with a stir bar, 120.0 mg (0.870 mmol) of K₂CO₃ and 95.2mg (0.48 mmol) S-3-bromopropyl ethanethioate was added. The mixture wasstirred while heated by 50° C. oil bath for 5 h. The reaction mixturesof the two reactions were combined, filtered and evaporated. Crudeproduct (168.4 mg) was obtained and co-evaporated with hexane for 3times to remove ACN. Silica gel column (1.25 g) was used to purify thecrude product. Yield 14% (2.6 mg). Structure of the product was provedby NMR and LC-MS

Synthesis of3-((3-(2,4-dichlorophenoxy)propyl)(prop-2-ynyl)amino)propane-1-thiol

A solution ofS-3-((3-(2,4-dichlorophenoxy)propyl)(prop-2-ynyl)amino)propylethanethioate (1.17 mg, 3.10 μmol) in 200 μL ACN was added into a 20 mLvial equipped with a stir bar, evaporated and then co-evaporated withMeOH for 3 times to remove ACN. MeOH/HCl (200 μL) was added into thevial and then the vial was heated by 85° C. oil bath for 6 h. Thereaction mixture was evaporated, co-evaporated sequentially by MeOH for3 times and ACN for 3 times, and then used in the next step withoutfurther purification.

Synthesis of MHI 148-clorgyline conjugate

MHI-148 (4.7 mg, 6.2 mmol) and EDC (1.5-2.4 mg, 7.8-12 mmol), followedby 1.5 mg of DMAP (12 mmol) were added into a 20 mL vial equipped with astir bar. ACN (400 μL) was added to make solution. The reaction mixtureof the previous step was transferred dropwise to the vial with 200 μLACN at room temperature. The reaction mixture was purified by HPLC(GRACE Davison Apollo C₈ 5u column, 250 mm×10 mm).

EXAMPLE 1 MAO A is Expressed in Human Glioma Tissues and Cells

The results show that there is significant staining of MAO A in GBMtissues while no detectable staining was observed in non-tumor braintissue (FIG. 2A). Based on morphology, the staining in GBM appears to beassociated with tumor cells. Human glioma cells, sensitive (U2515) andresistant (U251R), both expressed MAO A as shown by immunostaining (FIG.2B). These glioma cell cultures were then analyzedfor MAO activity (5)using serotonin as substrate (FIG. 2C) and showed that these gliomacells expressed MAO A catalytic activity; in contrast, normal controlastrocytes exhibited no detectable MAO A activity.

MAO A activity was also measured in the mouse glioma cell line, GL26;these tumor cells showed high levels of MAO A activity. Based on thisinformation, GL26 cells were chosen to evaluate the inhibitory effectsof different MAO inhibitors. Clorgyline and phenelzine inhibited MAO Aactivity with low IC₅₀ of 10⁻⁹ and 10⁻¹¹ M, respectively.Tranylcypromine and deprenyl inhibited MAO A activity with IC₅₀ value inlow micromolar range. This inhibition profile confirmed that indeed MAOA was present in GL26 cells. Taken together, these results showed thatMAO A is present in human glioma cells and human GBM tissue but theywere not detectable in normal astrocytes.

EXAMPLE II MAO A Inhibitor Clorgyline and NMI (Near-Infrared DyeConjugated MAO Inhibitor) Reduced Proliferation, Viability and Migrationof TMZ-Sensitive and Resistant Glioma Cells

MAO A inhibitors target MAO A expression in both central nervous systemand peripheral tissues (9). To target MAO A specifically to brain cancercells, the MAO inhibitor clorgyline was conjugated to a near infra red(NIR) tumor-specific dye, MHI-148, to produce the novel drug NMI (FIG.5A) that would preferentially accumulate in cancerous lesions. NMI hasbeen synthesized according to the procedure shown in FIG. 1.

To evaluate the cellular uptake of NMI in human glioma cells confocalmicroscopy was used. Image analysis of tumor cells treated with NMI (1μM, 5 μM) showed a significant dose-dependent, uptake compared tovehicle. This compound rapidly accumulated in U251R cells and localizedto the mitochondria, as determined by the co-localization of themitochondria-specific dye, MitoTracker Green (FIG. 5B). The inhibitoryeffect of NMI on MAO A activity was performed on GL26 mouse gliomacells, which has abundant MAO A activity. The results show that NMIinhibits MAO A with low micromolar IC₅₀ (FIG. 3). These results indicatethat NMI targeted specifically to glioma cell mitochondria and inhibitedMAO A activity in vitro.

Tumor recurrence is due to the development of TMZ resistance. Therefore,whether clorgyline and NMI is cytotoxic to TMZ-resistant glioma cellsusing resistant (U251R) glioma cells was investigated. TMZ-sensitiveglioma cells (U251S) were used for comparison. For TMZ-sensitive cells,U251S, clorgyline (10 μM) itself reduced the colony formation by 20%(p<0.05) and TMZ (15 μM) by 60%. Combined treatment of clorgyline withTMZ reduced it further to 65% (FIG. 4A). These results indicated thatclorgyline increased TMZ sensitivity in drug-sensitive glioma cells(U2515) in vitro.

Treatment of NMI alone in U251S cells at 5 μM and 10 μM reduced colonyformation by 60% and 90%, respectively (FIG. 5C (a)). Combine NMI (5 μM)and TMZ (15 μM) reduced colony formation by 90%. (FIG. 5C (a)). Theseresults indicated that NMI increased TMZ sensitivity in drug-sensitiveglioma cells (U251S) in vitro.

The effects of NMI on drug-resistant human glioma cell line, U251R werethen evaluated. TMZ (μM) has no effect as expected. NMI exhibited aconcentration dependent decrease in colony formation at 1, 5 and 10 μMby 20%, 40% and 80%, respectively (FIG. 5C (b)). NMI at 10 μM sensitizedthe TMZ-resistant cells to TMZ treatment and exhibited greater than a95% decrease in proliferation. (FIG. 5C (b)). These results showed thatNMI sensitized TMZ-resistant cells to TMZ. Clorgyline itself or thecombination with TMZ did not show any effect (FIG. 4B).

Cytotoxic effects of clorgyline and NMI on glioma cells were alsostudied using the MTS assay (FIG. 5D (a) and (b)); results demonstratedthat treatment with clorgyline produced dose response curves with 50%inhibitory concentrations (IC₅₀) of approximately 175 μM and 136 μM inU251S and U251R cells, respectively. By contrast, treatment with NMIinhibited cell viability with an IC₅₀ value of 5 μM in both cell lines,indicating 30 to 35 fold higher efficacy of NMI as compared toclorgyline. Thus NMI is an effective cytotoxic agent alone or incombination with TMZ in drug-resistant glioma tumor cells.

GBM is a highly invasive tumor; therefore a chemotherapeutic agent thataffects tumor cell migration and invasion would be very usefulclinically. The ability of NMI to inhibit migration of U251S and U251Rcells using the migration assay was studied. Cells were treated withclorgyline (10 μM) and NMI (1, 5 and 10 μM) for 20-24 hours dependingupon the time required for complete closure in vehicle treated cells.Clorgyline itself had no effect on the migration rate of human gliomacells (FIGS. 4C & D). However, NMI (5 μM) decreased migration rate by50% in sensitive cells (U2515) and by 30% in resistant cells (U251R)(FIG. 5E (a) and (b)). These results indicated that NMI is moreeffective in decreasing the migration rate in U251 TMZ-resistant cellscompared to clorgyline.

EXAMPLE III Genetically Modified MAO A Knockout (MAO A KO) AnimalsExhibited Increased Survival

It was determined that MAO A is expressed in human GBM tissues using IHC(FIG. 2B), in contrast to the little staining of MAO A in non-tumorbrain. These results showed higher expression of MAO A in glioma cellsand GBM tissues suggesting that MAO A may be involved in GBMprogression.

To examine the in vivo effects of MAO A on tumor growth, tumorprogression was analyzed in MAO A knockout mice (KO) in C57bl/6 mice.Mouse glioma cells (GL26) derived from C57bl/6 mice were used in thesestudies because these tumor cells showed high levels of MAO A activity(FIG. 2C). The tumor cells were labeled with luciferase and implantedintracranially into MAO A KO and WT C57bl/6 mice; luciferase imaging wasperformed on day 10. The results showed a 75% reduction in tumor burden(FIGS. 6A and B) and 17.6% (3 days) increase in survival (FIG. 6C).

MAO A activity in the tumor and surrounding tissue of the WT and AKOmice was measured at the end of the experiment. The results show thatMAO A KO had low MAO A activity in the tumor tissue as compared to WT.Interestingly, these results indicated that the surrounding tissue withno MAO A activity in KO mice may affect the activity of MAO A in tumortissue (FIG. 6D). Similar results were obtained with subcutaneousimplantations of GL26 tumor cells in MAO A KO and WT mice. These resultssuggested that a reduction of MAO A in the tumor and tumormicroenvironment decreased GBM progression.

EXAMPLE IV Clorgyline Inhibited the Growth of Human Tumor Cells in theSubcutaneous Xenograft Glioma Mouse Model

To determine the in vivo effects of clorgyline on tumor progression,U251S luciferase-labeled tumor cells were implanted subcutaneously intonude mice; when tumors were visible by imaging, clorgyline (30 mg/kg),dissolved in sterile water, was administered daily for 21 days. Thevehicle control group was treated with water. All mice in treated andcontrol groups showed similar changes in body weight that did not exceed10% of weight loss, indicating that this treatment regimen was welltolerated. Starting from day 28, the clorgyline-treated groups showed aconsistent decrease in tumor growth with 70% decrease at day 41 (FIGS.7A & 7B). These results demonstrated that inhibition of MAO A reducedtumor growth in vivo.

EXAMPLE V Clorgyline and NMI Increased the Survival Response, Alone orin Combination with TMZ in the Intracranial Mouse Tumor Model

To determine whether MAO A inhibitors were also effective againstTMZ-resistant glioma cells in vivo, human TMZ-resistant glioma cells(U251R), were implanted intracranially and imaged after 7 days. Micewere then grouped (n=4) and treated with: clorgyline, NMI, TMZ and/orcombination of TMZ and clorgyline or NMI. Drugs were administeredsubcutaneously daily for 21 days except for TMZ, which was administeredby oral gavage for 10 days at a dose of 1 mg/kg. Clorgyline was injectedat 10 mg/kg and NMI at 5 mg/kg. NIR imaging in vivo showed the specificuptake and accumulation of NMI in glioma cells. Bioluminescence ofluciferase-labeled cells as well as fluorescence of NMI was recordedafter 10 days of daily subcutaneous injection of NMI (5 mg/kg/day).Overlaying the NIR image with bioluminescence showed that NMI crossedthe blood brain barrier, and localized to the tumor site with nodetectable distribution throughout the body (FIG. 8A). Animals wereimaged on days 7, 14, 21, 24 (FIG. 8B). After 28 days (7 days afterimplantation and 21 days of treatment) treatment was stopped; tumorgrowth and survival was documented (FIGS. 8C and 8D). Survival datashowed that all animals in the vehicle group died by day 28, animalsfrom the TMZ and clorgyline groups died by 34 (p<0.05) and 36 days(p<0.05), respectively. The combination treatment of clorgyline with TMZincreased the survival to 40 days from the day of tumor implantation(p<0.05). Treatment with NMI alone exhibited increased survival by 12days (died by day 41, p<0.005); and combination of TMZ and NMI increasedthe survival even further by 16 days (died by day 44, p<0.005); theKaplan-Meier plot showing these data are presented in FIG. 4D. All pvalues are reported with respect to vehicle. All mice in treated andcontrol groups showed similar changes in body weight that did not exceed10% throughout the entire duration of experiment. These data indicatethat treatment with clorgyline or NMI alone, or NMI in combination withTMZ can delay tumor growth and significantly increase survival time.

EXAMPLE VI Clorgyline and NMI Reduced Proliferation and Angiogenesis andInduced the Innate Immune Response In Vivo

In order to identify potential mechanisms for increased survival ofclorgyline or NMI treated animals, tumor tissues were harvested atdeath, and analyzed for several characteristics including cellproliferation, microvessel density (MVD), inflammatory cellinfiltration, and secretion of growth factors. To determine whether cellproliferation was affected by MAO A inhibitors, tumor tissues werestained with Ki67. The results show that the number of positive cells(red precipitate) was decreased in clorgyline and NMI (p<0.05, FIG. 9C)treated animals, compared to vehicle-treated animals (FIG. 9A, row 1).Tissues were analyzed for matrix metalloproteinase 9 (MMP9), an enzymeresponsible for destruction of extracellular matrix and therebyincreasing tumor invasion (13, 14). The results (FIG. 9A, row 2)indicated more positive staining for MMP9 in vehicle tissues as comparedto clorgyline or NMI treated animals. These data suggest that MAOinhibitors reduced tumor cell invasiveness. Angiogenesis and bloodvessel density, critical for tumor progression (10), were assessed bystaining for CD31, endothelial cell marker. The results (FIG. 9A, row 3;FIG. 9C) showed that clorgyline (p<0.01) and NMI (p<0.05)-treatedanimals have significantly reduced MVD. These data demonstrated thatclorgyline and NMI significantly reduced proliferation, invasion andangiogenesis in tumors, thereby contributing to enhanced survival.

The innate immune response is an important contributor to the regulationtumor growth (11). Therefore, inflammatory cells in the tumors oftreated animals were analyzed. Tumor tissues were stained with F4/80,macrophage marker (FIG. 9B, row 1). The results showed a significantincrease in macrophages in MAO inhibitor-treated animals as compared tovehicle control (p<0.01, FIG. 9C). These data suggested that macrophagesmay be involved in delayed tumor progression. Inflammatory cytokines areresponsible for much of the activity attributed to macrophages. Todetermine whether the macrophages detected in tumor tissues wereproinflammatory, tissue specimens were stained for tumor necrosis factor(TNF)-α, a powerful proinflammatory growth factor. The staining resultsdemonstrated an increased TNF-α positive population in tumors from MAOinhibitor-treated animals (FIG. 9B, row 2). These results suggested thatMAO inhibitors upregulate the proinflammtory response, which correlateswith macrophage presence and decreased tumor progression (12).Transforming growth factor TGF β, an immune suppressive growth factor,was not affected by MAO A inhibitor treatment (FIG. 9B, row 3); TGF βstaining was similar in all three groups. This staining data of tumortissues provide compelling evidence that clorgyline and NMI inhibitorsalter the tumor environment to n reduce tumor growth in vivo.

EXAMPLE VII MAO A Inhibitors Increased Animal Survival in theIntracranial Mouse Model

The effects of other MAO A inhibitors on tumor progression wereexamined. Labeled mouse glioma cells (GL26) were implantedintracranially and imaged 7 days post implantation. Subsequently animalswere randomly grouped (n=5); drug treatment groups were TMZ (1 mg/kg),phenelzine (10 mg/kg), phenelzine+TMZ, and moclobemide (10 mg/kg).Phenelzine is an MAO A and B inhibitor; and Moclobemide is an MAO Aspecific reversible inhibitor. All drugs except for TMZ wereadministered daily subcutaneously; TMZ was administered orally. Survivaldata showed that all vehicle-treated animals died by day 12; TMZ andphenelzine (p<0.05) groups died by day 14. Phenelzine+TMZ (p<0.005) andmoclobemide (p<0.005)-treated mice died at day 14 and 16, respectively.The Kaplan-Meier plot showing these data are presented in FIG. 10A. Allp values are reported with respect to vehicle. These results showed thatphenelzine in combination with TMZ and moclobemide increased survival.

To better understand the role of MAO activity in tumor progression,brain tissues were harvested, and analyzed for MAO A activity.Phenelzine and moclobemide exhibited 87% and 62% reductions in enzymeactivity, respectively (FIG. 10B). These results indicated that theincrease in survival correlates with decreased MAO A activity.

EXAMPLE VIII Further Results Supporting the Unexpected Nature of thePresent Invention

FIG. 18 discloses data indicating that prostate cancer and glioma haveMAO A activity and can be treated with MAO I and MHI-clorgyline, whereaspancreatic cancer and lymphoma do not have MAO A activity, thus cannotbe treated by clorgyline and MHI-clorgyline.

FIG. 19 demonstrates that clorgyline and MHI-clorgyline both induce stemcell cytotoxicity and MHI-clorgyline is more effective than clorgylineby itself.

Although the present invention has been described in terms of specificexemplary embodiments and examples, it will be appreciated that theembodiments disclosed herein are for illustrative purposes only andvarious modifications and alterations might be made by those skilled inthe art without departing from the spirit and scope of the invention asset forth in the following claims.

REFERENCES

The following references are each relied upon and incorporated herein intheir entirety.

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1. A method of treating brain cancer, comprising: administering to apatient having brain cancer an effective amount of an MAO inhibitor. 2.The method of claim 1, wherein the brain cancer comprises aglioblastoma.
 3. The method of claim 2, wherein the glioblastoma is atemozolomide resistant glioblastoma.
 4. The method of claim 3, furthercomprising administering temozolomide in combination with the MAOinhibitor.
 5. The method of claim 4, wherein the temozolomide isadministered in combination with the MAO inhibitor.
 6. The method ofclaim 5, wherein the temozolomide and the MAO inhibitor is administeredsequentially.
 7. The method of claim 1, wherein the brain cancer isGlioblastoma multiforme.
 8. The method of claim 1, further comprisingadministering temozolomide in combination with the MAO inhibitor.
 9. Themethod of claim 1, wherein the MAO inhibitor is selected from the groupconsisting of

and salts thereof.
 10. The method of claim 1, wherein the MAO inhibitoris selected from the group consisting of


11. The method of claim 9, wherein said monoamine oxidase is covalentlylinked to a near infrared dye via a linker.
 12. The method of claim 9,wherein the near infrared dye comprises a polyene functional groups. 13.The method of claim 12, wherein the near infrared dye is selected fromthe group consisting of IR-783, IR-780, IR-786 and MHI-148
 14. Themethod of claim 13, wherein the MAO inhibitor is selected from the groupconsisting of NMI, MHI-moclobemide, MHI-phenelzine, MHI-tranylcypromine,MHI-pargyline, MHI-clorgyline, MHI-MAOIs, and NIR-MAOIs.
 15. The methodof claim 12, wherein the linker has the following formula:

wherein M₁ is O or S; wherein at least two of X, Y, and Z participate inbonds to groups A and B which may be unsaturated groups, aromaticgroups, or both, said groups A and B may further be connected toadditional carbon, oxygen or nitrogen atoms; and wherein any of X, Y,and Z not bonded to groups A or B is substituted with hydrogen or loweraliphatic groups having 1-6 carbon atoms.
 16. The method of claim 1,wherein the MAO inhibitor has the formula:


17. The method of claim 1, wherein the MAO inhibitor is selected fromthe group consisting of

and salts, carboxylic acids or esters thereof.
 19. The method of claim1, further comprising concurrently or sequentially administering to thepatient one or more additional treatments for brain cancer, wherein theone or more additional treatments include surgery, radiation andchemotherapy.
 19. A compound comprising a salt of

or a carboxylic acid or ester analog thereof.
 20. A pharmaceuticalcomposition comprising the compound of claim
 19. 21. A method oftreating brain cancer, comprising: Administering to a person in needthereof an effective amount of the pharmaceutical composition of claim20.